The present invention involves novel cytokines, in particular Gro genes and proteins, which were derived from the cDNAs of induced peripheral blood cells, for example monocytes. Therefore, the following discussion presents the background on cytokines, monocytes and the Gro genes and proteins.
A. Cytokines:
Cytokines are small molecular weight proteins that have a broad range of cell regulatory activity both in vitro and in vivo. (For background information, see Balkwill, F. R., et al., 1989, Immun. Today, 10:299). Some cytokines possess growth factor activity. Examples of cytokines which can inhibit cell growth or directly kill cells are interferons (IFNs), tumor necrosis factor (TNF), lymphotoxin (LT), the interleukin 1s (IL-1s) and transforming growth factor .beta. (TGF-.beta.). Most cytokines act on B or T cells at some stage in their response. An example of the physiological effect of cytokines can be seen in the case of cytokines which regulate the inflammatory response by varying the T and B lymphocyte activation, chemotaxis, eicosanoid secretion and collagen production. Differential expression of these inflammatory mediators may influence the perpetuation of chronic inflammation and fibrotic manifestations.
B. Monocytes:
Monocytes are directed to sites of infection by chemotactic factors released as a result of inflammation or infection (Sporn, S. A., et al., 1990, J. of Immun., 144:4434-4441). The migration of monocytes into the tissue involves interaction with vascular endothelium and the subsequent migration of the monocytes through the underlying basement membrane, during which the monocytes come into close contact with extracellular matrix components and connective tissue cells (Harlan, J. M, 1985, Blood, 65:513; Wilkinson, P. C., et al., 1979, Curr. Top Pathol., 68:47).
Thorens, B., et al., 1987, Cell, 48:671 reported that adherence might be important in transcriptional expression of macrophage mediators of inflammation and that adherence to different matrices could result in preferential gene induction. Further, there is evidence for participation of cell adhesion molecules in developmental interaction between epithelial and mesenchymal cells influencing cell differentiation (Sanders, E. J., 1988, Biochem. Cell Biol., 66:530). It has also been shown that within 30 minutes of monocyte adherence to plastic, a complex set of regulatory events is initiated as defined by rapid changes of mRNA levels of several proto-oncogenes and inflammatory mediators (Haskill, S., et al., 1988, J. of Immunol., 140:1690). IL-1.beta., TNF-.alpha. and c-fos are rapidly elevated, whereas CSF-1 steady state mRNA levels increase by 90 minutes. In contrast, expression of c-fms and lysozyme is rapidly down-regulated. These genes are modulated by adherence to different biologically relevant substrates (Eierman, D. F., 1989, J. of Immunol., 142:1970-1970).
Although high steady state mRNA levels of important mediators of inflammation are rapidly induced by adherence, adherence by itself is insufficient to cause efficient translation and secretion of IL-1.beta., TNF-.alpha., or CSF-1 (Haskill, S., et al., supra). Activation by a second signal, such as bacterial endotoxin, is required for the secretion of all three gene products. Thus, it is clear that signals derived from the act of adherence are likely to play a significant role in the activation and differentiation of monocytes allowing them to respond to infection and to influence the local tissue environment (Sporn, S. A, supra). Recent studies indicate that adherence leads to a general activation of numerous genes involved in the early defense response and whose expression may be regulated by selective tissue/extracellular matrix interactions. Id.
C. Gro Genes/Proteins:
Adherence of monocytes results in the rapid induction of high levels of mostly transient mRNAs for various novel inflammatory mediator genes (Spom, S. A., et al., supra). One of these novel clones, Gro .alpha., had a predicted amino acid sequence similar to that reported for the original Gro gene product. Mapping studies of Gro (Anisowicz, A., et al., 1988, PNAS (USA), 85:9645-9649 and Richmond, A., et al., 1988, EMBO J., 7:2025-2033) identified a unique Gro site at chromosome 4q21. The Gro gene belongs to a gene super-family which encodes a set of related cytokines that includes NAP-1/IL-8 (Matsushima, K., et al., 1988, J. Exp. Med., 167:1883-1893; Schmid, J., et al., 1987, J. of Immunol., 139:250-256; Peveru, P., et al., 1988, J. Exp. Med., 167:1547-1559), and platelet basic protein (PBP). PBP is the precursor of connective tissue activating protein III (CTAP III), .beta.-thromboglobulin (Castor, C. W., et al., 1983, PNAS (USA), 80:76-769), platelet factor 4 (PF4) (Deuel, T. F, et al. 1977, PNAS (USA), 74:2256-2258), .gamma.-interferon-inducible peptide (.gamma.IP-10) (Luster, A. D., et al., 1985, Nature (London), 315:672-676), and macrophage inflammatory protein 2 (MIP-2) (Wolpe, S. D., et al., 1989, PNAS (USA), 86:612-616).
Gro was initially identified by its constitutive over-expression in spontaneously transformed Chinese hamster fibroblasts (Anisowicz, A., et al., 1987, PNAS (USA), 84:7188-7192). A related gene was identified in v-src transformed chicken cells (Sugano, S., et al., 1987, Cell, 49:321-328; Bedard, P. A., et al., 1987, PNAS (USA), 84:6715-6719). In expression studies with normal fibroblasts, Gro showed early response kinetics similar to c-fos, leading to the name Gro (growth regulated) (Anisowicz, A., et al., 1987, supra). Later, a protein with melanoma stimulating activity (MGSA) (Richmond, A., et al., supra) was shown to be encoded by Gro, and sequence similarity was reported with the murine early response gene KC (Oquendo, P., et al., 1989, J. Biol. Chem., 264:4133-4137).
Preliminary studies showed that the Gro ax gene was expressed in active ulcerative colitis disease, but not in the inactive tissue. (Isaacs, K., et al., "Profiles of cytokine activation in inflammatory bowel disease tissue: measurement of cDNA amplification", American Gastroenterological Assoc. & American Assoc. for the Study of Liver Diseases, May 13-16, 1990, Texas (Abstract)). On the other hand, disparity in expression of the Gro .alpha. gene was less in the case of active versus inactive tissues from Crohn's disease. The expression of the Gro .alpha. gene in active intestinal inflammation suggests a role of these cytokines in the pathogenesis of inflammatory bowel disease.
A cDNA designated MAD-2 was shown at a 1988 RES Conference. (Sporn, S. et al., "Isolation of Adherence Specific cDNA Clones from a Monocyte cDNA Library" Society for Leukocyte Biology (RES), Washington, D.C., Oct. 27-30, 1988; and in Sporn, S. A. et al. J. of Immunol, 1990, 144:4434.) MAD-2 was isolated by differential hybridization from a cDNA library which was prepared in the bacterial expression vector .lambda.gt10 by using total RNA from human blood monocytes adhered to plastic for 30 minutes at 37.degree. C. Sporn, S. A. et al., J. of Immunol, 1990, supra. MAD-2 and human Gro were believed to be separate gene products based on Southern blots analysis and differences in the amino acid sequences in the carboxyl-terminus amino acids. Id. at 4440. At the Cytokine Workshop of Dec. 13, 1989, Hilton Head, Georgia, Ruth Sager presented the amino acid sequences of both Gro .beta. and .gamma..
Recently, two cDNAs for the human homologs of murine MIP-2 had been cloned from a library prepared from phorbol myristate acetate (PMA)--treated and lipopolysaccharide (LPS)--stimulated U937 cells Tekamp-Olson, P. et al., 1990, J. Exp. Med., 172:911. These cDNAs, designated MIP-2.gamma. and MIP-2.beta. are closely homologous to the cDNAs disclosed in the pending patent application.